The disclosure generally relates to methods for amplifying the Raman signal of surface enhanced Raman scattering (SERS) nanoparticles.
Surface enhanced Raman scattering allows for the detection of molecules attached to the surface of a single metallic nanoparticle, typically a gold or silver nanoparticle. Existing SERS nanoparticles, also referred to as nanotags, generally include the metallic nanoparticle having a reporter molecule in close proximity thereto (typically less than 50 angstroms), which produces a strong Raman signal due to a surface enhanced effect. Bringing reporter molecules in close proximity to the metal surfaces is typically achieved by adsorption of the Raman-active molecule onto suitably roughened metal nanoparticles, e.g., gold, silver, copper, or other free electron metals. The characteristic signal of the reporter molecule is used to determine the presence and amount of the SERS nanoparticles. Consequently, SERS nanoparticles have utility as spectroscopic and optical tags and are often used in assays.
SERS nanoparticles are somewhat versatile and can be functionalized with biological molecules (e.g., antibodies, DNA, and the like) so that they specifically bind to one kind of target (e.g., specific types of bacteria, viruses, spores, proteins, DNA, and the like). For example, SERS nanoparticles can be used in immunoassays when conjugated to an antibody against a target of interest. If the target of interest is immobilized on a solid support, then the interaction between a single target and a single nanoparticle-bound antibody could be detected by searching the unique Raman spectrum for the Raman-active reporter molecule. Furthermore, because a single Raman spectrum (from 100 to 3500 cm−1) can detect many different Raman-active molecules, SERS nanoparticles can often be used in multiplexed assay formats.
SERS is believed to occur primarily as a result of surface plasmon resonances in the metal nanoparticle that enhance the local intensity of the light, and formation and subsequent transitions of charge-transfer complexes between the metal surface and the Raman-active reporter molecule.
Protocols for producing SERS nanoparticles from colloidal solutions of metallic nanoparticles present formidable practical problems. For example, metal nanoparticles are exceedingly sensitive to aggregation in aqueous solution; once aggregated, re-dispersion is generally impossible. In addition, the chemical compositions of some Raman-active reporter molecules are incompatible with the chemistries used to attach other molecules (such as proteins) to the metal nanoparticles. This restricts the choices of Raman-active reporter molecules, attachment chemistries, and other molecules to be attached to the metal nanoparticle.
By design, the Raman spectroscopic signal from a SERS nanotags is dominated by the surface-enhanced Raman spectrum of the attached reporter molecule. Other attached moieties, such as the recognition element (e.g., antibody), or encapsulating shells (e.g., glass), do not contribute to the observed Raman spectrum because they are either Raman inactive, their Raman signal is not surface-enhanced due to the particular bonding geometry, their distance from the metal surface is too great, or other reasons.
Current processes for making the SERS nanoparticles are numerous. One method as described in U.S. Pat. No. 6,514,767 to Natan generally follows a synthetic pathway as outlined in the schematic provided in prior art FIG. 1. The synthetic pathway generally starts with a colloidal solution, e.g., HAuCl4 (i.e., gold chloride) colloidal solution, and a reducing agent that results in the precipitation of gold nanoparticles having average diameters of about 60 nm. The reducing agent is composed of a single reductant, typically a citrate salt, e.g., sodium citrate, to reduce the gold and form a stable colloid. The resulting colloid is generally red in color and exhibits an absorption peak (λmax) at about 530 nm. An amino-based silane is then added to form vitreophilic surfaces capable of accepting the desired tag or reporter molecules. Next, a silicate is added, which polymerizes onto the “tagged” gold nanoparticle surface. The thickness of the silicate layer is typically on the order of a few nanometers. A thicker shell can be formed if desired using tetraethylorthosilicate (TEOS). During or after this step, the glass-coated nanoparticle can also be functionalized such as with 3-mercaptopropyltrimethoxysilane (MPTMS) or 3-aminopropyltrimethoxysilane (APTMS) to form SERS nanoparticles with corresponding end groups having sulfhydryl or amino functionalization. Optionally, a second silane-coupling agent can be used depending on the polarity of the solvent in which the particles are to be dispersed. In this manner, the nanoparticles can be dispersed in a low polarity solvent if desired for the particular application. Target molecules with the appropriate linker chemistry are reacted with the end groups to provide the tagged SERS nanoparticles. For example, antibody conjugated SERS nanoparticles can be formed.
Other methods for producing SERS-active nanotags provide different particle architectures. For example, Nie and Doering as described in PCT Application No. WO 2005/062741 used organic dyes adsorbed to a metallic core, and also encapsulated the resulting particle with a glass shell. In U.S. Patent Publication No. US20050089901 A1 to Porter, a tag is built from a metal nanoparticle core, and in this case, the Raman-active molecule is specifically chosen to have a reactive end that binds to the metal nanoparticle surface and another part that acts as a linker to the biological attachment part, so that the overall SERS nanotags does not have a glass shell. In US Published Patent Application No. 2005/0158877 A1 to Wang et al., analyte analogues are first attached to a metallic particle surface. Then the metallic colloidal solution is mixed with an antibody solution. Each antibody molecule will bind with two analyte analogue molecules, thus causing the metallic particles to aggregate and form a cluster structure for SERS signal amplification. In the presence of analyte, the antibody molecule reacts with the analyte molecule and the formation of the cluster structure is inhibited, which results in a decrease of the Raman signal. Thus the presence and amount of the analyte can be inferred from the intensity variation of the Raman signals. In each instance, each SERS particle has both the Raman dye and the antibody.
During the preparation of SERS nanotags, starting with a colloidal solution of metallic nanoparticles, it is often observed that the Raman signal of the adsorbed reporter molecule is substantially enhanced when the metallic nanoparticles begin to aggregate due to addition of an excess amount of the reporter molecule, or an excess of another adsorbate along with the reporter molecule. However, gross aggregation prevents further processing of the SERS nanotags, since they begin to form precipitates that are not re-dispersible, and are often not amenable to further modification such as antibody attachment. Thus, protocols for preparation of SERS nanotags are designed to eliminate or minimize aggregation.
Regardless of the method used for producing the SERS active nanoparticles, there remains a need in the art for amplification of the Raman signal to improve the detection limits especially with regard to the SERS nanoparticles.